Project Summary Despite their potential to suppress tumor growth, natural killer (NK) cells do not have significant activity against solid tumors due to limited activity in the tumor microenvironment (TME). We seek to identify the molecular basis for this limitation and to develop a strategy for improving NK cell?mediated antitumor activity. An underlying premise for our approach is that some established cytotoxic chemotherapies enhance tumor cell immunogenicity by promoting autophagy, immune stimulating damage-associated molecular pattern (DAMP) molecules, cytokines/chemokines, and immunogenic cell death. These immune stimulatory signals enhancing the antitumor immune response improving therapeutic outcomes. These effects can be further enhanced by combining chemotherapy with an immunotherapy designed to enhance effector cell (CD8 T cell or NK cell) antitumor activity. It is with this strategy in mind that we have combined the chemotherapies doxorubicin or paclitaxel, with our novel immunotherapy that depletes the epigenetic regulator the nucleosome remodeling factor (NURF). Our preliminary studies demonstrated that this combinatorial treatment enhances the cytotoxic effects of doxorubicin (increased DNA damage, autophagy and reduced tumor cell growth), in breast cancer cells. The combined treatment improves tumor cell immunogenicity as measured by enhanced NK cell killing and reduced tumor growth in animal models. Similar increases in autophagy, reductions in tumor cell growth, and enhanced tumor cell immunogenicity were observed using a novel small molecule inhibitor of NURF (AU1), demonstrating that NURF can be targeted therapeutically. It is the objective of this proposal to characterize how pharmacological NURF inhibition enhances sensitivity to chemotherapy, and if these effects improve therapeutic outcomes using preclinical models of metastatic breast cancer. To test this hypothesis we will complete two Specific Aims. In Aim1 we will develop strategies to improve NK cell antitumor activity. Towards this end NURF depletion will be combined with chemotherapies to discover NK cell stimulating combinations. Promising combinations will be studied in vivo for effects on NK cell activation, maturation, proliferation (intra-tumor and peripheral), homing, and NK cell-mediated antitumor activity (in vivo and ex vivo). In Aim2 we will determine how NURF regulates breast cancer cell sensitivity to doxorubicin (and other chemotherapies as identified from Aim1). These studies will focus on therapy induced DAMP and cytokine/chemokine production, and how that could be regulated by increases in autophagy. New pharmacologic inhibitors to NURF will be used to repeat key observations from Aim1 and 2 to determine its therapeutic potential. Completing these Aims will discover how therapy treated tumors stimulate NK cell antitumor activity, further characterizing chromatin remodeling as a novel regulator of NK cell-mediated antitumor immunity and leveraging these discoveries to improve existing NK cell stimulating chemotherapies.